Influence of Etherel and Gibberellic Acid on Carbon Metabolism, Growth, and Essential Oil Accumulation in Spearmint (Mentha Spicata)

Changes in growth parameters and ¹⁴CO₂ and [U-¹⁴C]-sucrose incorporation into the primary metabolic pools and essential oil were investigated in leaves and stems of M. spicata treated with etherel and gibberellic acid (GA). Compared to the control, GA and etherel treatments induced significant phenotypic changes and a decrease in chlorophyll content, CO₂ exchange rate, and stomatal conductance. Treatment with etherel led to increased total incorporation of ¹⁴CO₂ into the leaves wheras total incorporation from ¹⁴C sucrose was decreased. When ¹⁴CO₂ was fed, the incorporation into the ethanol soluble fraction, sugars, organic acids, and essential oil was significantly higher in etherel treated leaves than in the control. However, [U-¹⁴C]-sucrose feeding led to decreased label incorporation in the ethanol-soluble fraction, sugars, organic acids, and essential oils compared to the control. When ¹⁴CO₂ was fed to GA treated leaves, label incorporation in ethanol-insoluble fraction, sugars, and oils was significantly higher than in the control. In contrast, when [U-¹⁴C]-sucrose was fed the incorporation in the ethanol soluble fraction, sugars, organic acids, and oil was significantly lower than in the control. Hence the hormone treatment induces a differential utilization of precursors for oil biosynthesis and accumulation and differences in partitioning of label between leaf and stem. Etherel and GA influence the partitioning of primary photosynthetic metabolites and thus modify plant growth and essential oil accumulation. This revised version was published online in June 2006 with corrections to the Cover Date.     

Partitioning of Photosynthetically Fixed 14CO2 Into Oil and Curcumin Accumulation in Curcuma Longa Grown Under Iron Deficiency

Changes in leaf growth, photosynthetic efficiency, and incorporation pattern of photosynthetically fixed ¹⁴CO₂ in leaves 1 and 2 from plant apex, in roots, and rhizome induced in Curcuma by growing in a solution culture at Fe concentration of 0 and 5.6 g m^–3 were studied. ¹⁴C was incorporated into primary metabolites (sugars, amino acids, and organic acids) and secondary metabolites (essential oil and curcumin). Fe deficiency resulted in a decrease in leaf area, its fresh and dry mass, chlorophyll (Chl) content, and CO₂ exchange rate at all leaf positions. The rate of ¹⁴CO₂ fixation declined with leaf position, maximum being in the youngest leaf. Fe deficiency resulted in higher accumulation of sugars, amino acids, and organic acids in leaves at both positions. This is due to poor translocation of metabolites. Roots and rhizomes of Fe-deficient plants had lower concentrations of total photosynthate, sugars, and amino acids whereas organic acid concentration was higher in rhizomes. ¹⁴CO₂ incorporation in essential oil was lower in the youngest leaf, as well as incorporation in curcumin content in rhizome. Fe deficiency influenced leaf area, its fresh and dry masses, CO₂ exchange rate, and oil and curcumin accumulation by affecting translocation of assimilated photosynthates.     

Partitioning of 14C-Photosynthate of Leaves in Roots,Rhizome, and in Essential Oil and Curcumin in Turmeric (Curcuma longa L.)

Incorporation of photosynthetically fixed ¹⁴C was studied at different time intervals of 12, 24, and 36 h in various plant parts—leaf 1 to 4 from apex, roots, and rhizome—into primary metabolites—sugars, amino acids, and organic acids, and secondary metabolites—essential oil and curcumin—in turmeric. The youngest leaves were most active in fixing ¹⁴C at 24 h. Fixation capacity into primary metabolites decreased with leaf position and time. The primary metabolite levels in leaves were maximal in sugars and organic acids and lowest in amino acids. Roots as well as rhizome received maximum photoassimilate from leaves at 24 h; this declined with time. The maximum metabolite concentrations in the roots and rhizome were high in sugars and organic acids and least in amino acids. ¹⁴C incorporation into oil in leaf and into curcumin in rhizome was maximal at 24 h and declined with time. These studies highlight importance of time-dependent translocation of ¹⁴C-primary metabolites from leaves to roots and rhizome and their subsequent biosynthesis into secondary metabolite, curcumin, in rhizome. This might be one of factors regulating the secondary metabolite accumulation and rhizome development.     

Utilization of exogenously supplied 14C-saccharose into primary metabolites and alkaloid production in Catharanthus roseus

Partitioning of exogenously supplied U-¹⁴C-saccharose into primary metabolic pool as sugars, amino acids, and organic acids was analyzed and simultaneous utilization for production of alkaloid by leaf, stem, and root in twigs and rooted plants of Catharanthus roseus grown in hydroponic culture medium was determined. Twigs revealed comparable distribution of total ¹⁴C label in leaf and stem. Stems contained significantly higher ¹⁴C label in sugar fraction and in alkaloids [47 kBq kg⁻¹(DM)] than leaf. In rooted plants, label in ¹⁴C in metabolic fractions in root such as ethanol-soluble, ethanol-insoluble, and chloroform-soluble fractions and in components such as sugars, amino acids, and organic acids were significantly higher than in stems and leaves. This was related with significantly higher content of ¹⁴C in alkaloids in stems and leaves. ¹⁴C contents in sugars, amino acids, and organic acids increased from leaf to stem and roots. Roots are the major accumulators of metabolites accompanied by higher biosynthetic utilization for alkaloid accumulation.     

Boron Deficiency Induced Changes in Translocation of 14CO2-Photosynthate Into Primary Metabolites in Relation to Essential Oil and Curcumin Accumulation in Turmeric (Curcuma longa L.)

Changes in leaf growth, net photosynthetic rate (P _N), incorporation pattern of photosynthetically fixed ¹⁴CO₂ in leaves 1–4 from top, roots, and rhizome, and in essential oil and curcumin contents were studied in turmeric plants grown in nutrient solution at boron (B) concentrations of 0 and 0.5 g m^-3. B deficiency resulted in decrease in leaf area, fresh and dry mass, chlorophyll (Chl) content, and P _N and total ¹⁴CO₂ incorporated at all leaf positions, the maximum effect being in young growing leaves. The incorporation of ¹⁴CO₂ declined with leaf position being maximal in the youngest leaf. B deficiency resulted in reduced accumulation of sugars, amino acids, and organic acids at all leaf positions. Translocation of the metabolites towards rhizome and roots decreased. In rhizome, the amount of amino acids increased but content of organic acids did not show any change, whereas in roots there was decrease in contents of these metabolites as a result of B deficiency. Photoassimilate partitioning to essential oil in leaf and to curcumin in rhizome decreased. Although the curcumin content of rhizome increased due to B deficiency, the overall rhizome yield and curcumin yield decreased. The influence of B deficiency on leaf area, fresh and dry masses, CO₂ exchange rate, oil content, and rhizome and curcumin yields can be ascribed to reduced photosynthate formation and translocation.     

The Substrate Utilization and Concentration of 14C Photosynthates in Citronella under Fe Deficiency

Changes in the utilization pattern of primary substrate, viz. [U-14C] acetate, 14CO2 and [U-14C] saccharose, and the contents of 14C fixation products in photosynthetic metabolites (sugars, amino acids, and organic acids) were determined in Fe-deficient citronella in relation to the essential oil accumulation. There was an overall decrease in photosynthetic efficiency of the Fe-deficient plants as evidenced by lower levels of incorporation into the sugar fraction and essential oil after 14CO2 had been supplied. When acetate and saccharose were fed to the Fe-deficient plants, despite a higher incorporation of label into sugars, amino acids, and organic acids, there was a lower incorporation of these metabolites into essential oils than in control plants. Thus, the availability of precursors and the translocation to a site of synthesis/accumulation, severely affected by Fe deficiency, is equally important for the essential oil biosynthesis in citronella.     

Relationship between photosynthetic carbon metabolism and essential oil biogenesis in peppermint under Mn-stress

Changes in growth and yield parameters, and ¹⁴CO₂ and (U-¹⁴C)sucrose incorporation into the primary metabolic pool, and essentialoil have been investigated under Mn-deficiency and subsequentrecovery in Mentha piperita, grown in solution culture. UnderMn-deficiency, CO₂ exchange rate, total chlorophyll, total assimilatoryarea, plant dry weight, and essential oil yield were significantlyreduced, whereas chlorophyll a/b ratio, leaf area ratio andleaf stem ratio significantly increased. In leaves of Mn-deficientplants, ¹⁴CO₂ incorporation into the primary metabolic pool(ethanol-soluble and -insoluble) and essential oil were significantlylower, whereas (U-¹⁴C) sucrose incorporation into these componentswas significantly higher as compared to the control. Among theprimary metabolites, the label was maximum in sugars, followedby organic acids and amino acids. A higher label in these metaboliteswas, in general, observed in stems of Mn-deficient plants ascompared to the control. Mn-deficient plants supplied with completenutrient medium for 3 weeks exhibited partial recovery in growthand yield parameters, and essential oil biogenesis. Thus, underMn-deficiency and subsequent recovery, the levels of primaryphotosynthetic metabolites and their partitioning between leafand stem significantly influence essential oil biogenesis. Key words: Mentha piperita, Mn-stress, ¹⁴CO₂ and [U-¹⁴C] sucrose incorporation, oil accumulation, primary photosynthetic metabolites     

Influence of gibberellic acid on <Superscript>14</Superscript>CO<Subscript>2</Subscript> metabolism,growth, and production of alkaloids in <Emphasis Type="Italic">Catharanthus roseus</Emphasis>

Changes in growth parameters, carbon assimilation efficiency, and utilization of ¹⁴CO₂ assimilate into alkaloids in plant parts were investigated at whole plant level by treatment of Catharanthus roseus with gibberellic acid (GA). Application of GA (1 000 g m⁻³) resulted in changes in leaf morphology, increase in stem elongation, leaf and internode length, plant height, and decrease in biomass content. Phenotypic changes were accompanied by decrease in contents of chlorophylls and in photosynthetic capacity. GA application resulted in higher % of total alkaloids accumulated in leaf, stem, and root. GA treatment produced negative phenotypic response in total biomass production but positive response in content of total alkaloids in leaf, stem, and roots. ¹⁴C assimilate partitioning revealed that ¹⁴C distribution in leaf, stem, and root of treated plants was higher than in untreated and variations were observed in contents of metabolites as sugars, amino acids, and organic acids. Capacity to utilize current fixed ¹⁴C derived assimilates for alkaloid production was high in leaves but low in roots of treated plants despite higher content of ¹⁴C metabolites such as sugars, amino acids, and organic acids. In spite of higher availability of metabolites, their utilization into alkaloid production is low in GA-treated roots.     

14C-metabolism during callus induction in tobacco

Tobacco pith-phloem explants and callus were incubated in ¹⁴C-glucose, ¹⁴C-acetate or ¹⁴C-bicarbonate on different days in culture in the dark. ¹⁴CO₂ production and ¹⁴C incorporation into ethanol-insoluble components were generally greater in the subcultured callus than in the pith-phloem explants during days 0 to 5 in culture. Greatest radioactivity from all substrates was in the ethanol-soluble portion, which was further fractionated into lipids, amino acids, sugars and organic acids. Although incorporation into the different fractions varied with the substrate, the patterns of labelling were relatively similar in the two tissues. The greater wound metabolism in the subcultured callus in comparison to the pith-phloem explant during the induction phase of callus formation was correlated with the earlier visible initiation of cell proliferation in the subcultured tissue.     

Utilization of Photosynthetically Fixed 14CO2 into Alkaloids in Relation to Primary Metabolites in Developing Leaves of Catharanthus roseus

Partitioning of current photosynthates towards primary metabolites and its simultaneous incorporation in leaf alkaloids was investigated in developing leaves of medicinally important Catharanthus roseus. Of the total ¹⁴CO₂ assimilated, the leaves at positions 1–6 fixed 8, 22, 25, 19, 13, and 8 %, respectively, and stem 3 %. Leaf fresh mass, chlorophyll content, and CO₂ exchange rate increased up to the third leaf. The total alkaloid content was highest in young actively growing leaves, which declined with age. Total ¹⁴C fixed and its content in ethanol soluble fraction increased up to the third leaf and then declined. The ¹⁴C content in primary metabolites such as sugars and organic acids was also highest in the 3^rd leaf. The utilization of ¹⁴C assimilates into alkaloids was maximum in youngest leaf which declined with leaf age. Hence the capacity to synthesize alkaloids was highest in young growing leaves and metabolites from photosynthetic pathway were most efficiently utilized and incorporated into alkaloid biosynthetic pathway by young growing leaves.     

14C-Metabolism in cultured cotyledon explants of radiata pine

Excised cotyledons of radiata pine ( Pinus radiata D. Don), cultured under shootforming (plus cytokinin) and elongating (minus cytokinin) conditions, were incubated in ¹⁴C-glucose, ¹⁴C-acetate or ¹⁴C-bicarbonate at different stages of growth and differentiation. ¹⁴CO₂ was produced when the cotyledons were fed ¹⁴C-glucose and ¹⁴C-acetate (no measurement was made for ¹⁴C-bicarbonate feeding). Label from these precursors was incorporated into ethanol-soluble and -insoluble fractions. The largest percentage of radioactivity was associated with the ethanol-soluble portion, which was further fractionated into lipids, amino acids, organic acids and sugars. The amount of label and the pattern of labelling associated with each of the above classes of metabolites varied with time in culture and morphogenetic behaviour of the cotyledons. In general, there was a tendency towards a high rate of incorporation of label in elongating cotyledons during the period of rapid elongation. On the other hand, a high rate of incorporation of label in shoot-forming cotyledons coincided with the period of meristematic tissue formation. The data obtained support the hypothesis that organized development in vitro involves a shift in metabolism, which precedes and is coincident with the initiation of the process.     

Protein synthesis and uric acid excretion in the absence of essential amino acids in the silkworm,Bombyx mori

Effects of omission of single essential amino acids in the diet on incorporation of [¹⁴C]-labelled amino acid into tissue protein and on excretion of uric acid in the silkworm, Bombyx mori, were investigated. When larvae were reared on the diet deficient in isoleucine, the [¹⁴C]-leucine incorporated into tissue protein was less than that incorporated in larvae reared on the complete diet. This decrease of incorporation was also observed when any one of ten essential amino acids were omitted singly. This was accompanied by an enhanced excretion of uric acid by the larvae.     

METABOLISM OF ACETATE-1-14C IN EXCISED LEAVES FROM POTASSIUM DEFICIENT RICE SEEDLINGS

Excised leaves from one-month old rice seedlings grown in potassiumdeficient nutrient solution were fed with acetate-l-¹⁴C for20 to 180 min. Rate of respiration of those leaves was significantlyhigher than that of control leaves supplied with sufficientpotassium. Incorporation of UC into organic acid and sugar fractionswere greater in potassium deficient leaves than in normal leaves.The reverse was true as for the incorporation into amino acidfraction. In comparison with normal leaves, potassium deficientleaves acquired much ¹⁴C in succinic acid and glutamine. Therewere distinct differences in the labeling pattern of citrateand in the order of magnitude of specific activities among theorganic acids included in the TCA cycle. From these results,it was assumed that the increase of respiratory rate causedby the potassium deficiency was related to accelerated operationof the TCA cycle. The decreased rate of conversion of organicacids to amino acids and the accumulation of glutamine werealso concluded. (Received April 13, 1967; )     

Accumulation of low molecular weight solutes in water-stressed tropical legumes

Fourteen species of tropical legumes, representing 10 genera, were subjected to water stress. Youngest fully expanded leaves of stressed and unstressed plants were analysed for inorganic ions, sugars, inositols, organic acids, betaines and amino acids. The major compounds which accumulated with water stress were O-methyl-inositols (14 species), 2-methyl-2,3,4-trihydroxybutanoic acid-1,4-lactone (10 species) and proline (9 species). Concentrations of inorganic ions, sugars and organic acids decreased or were unchanged in the majority of the stressed species. The betaines, glycinebetaine, trigonelline and stachydrine were detected in low concentrations in most of the legumes but did not accumulate to any degree during water stress. All the legumes which tolerated low leaf water potentials accumulated the O-methyl-inositol, pinitol. The other species, with the exception of Siratro, contained ononitol or O-methyl-scyllo-inositol but no pinitol. It is suggested that pinitol accumulation may indicate a legume able to tolerate low leaf water potentials.     

C-Photosynthate Partitioning and Translocation in Soybeans during Reproductive Development

Partitioning and translocation of ¹⁴C-photosynthates were examined during flowering and seed maturation in soybean (Glycine max [L.]Merr.) plants to quantify allocation to sugars, amino acids, organic acids, and starch and to study transport of C and N from leaves to reproductive sinks. The trifoliolate leaf at the eighth node was exposed to steady state levels of ¹⁴CO₂ for 2 hours, followed by immediate extraction and identification of radioactive assimilates in the fed leaf blade, tissues of the transport path (e.g. petiole and stem), and fruits if they were present. About one-third of the total ¹⁴C recovered from the leaf blades was in starch until late pod-filling, after which the proportion dropped to 16%. Sugars comprised 70% to 86% of the recovered ¹⁴C from soluble assimilates of the source leaf, with highest proportions occurring during late flowering and early pod-filling. Amino acids accounted for 8% to 17% of the ¹⁴C recovered from the soluble fraction, and were most evident during early flowering and mid to late pod-filling. The ¹⁴C-organic acids comprised from 3% to 14% of the soluble ¹⁴C-assimilates in leaves. Petioles consistently contained a higher percentage of recovered radioactivity in sugars (87-97%) and a lower percentage in amino acids (3-12%) than did leaf blades. ¹⁴C-Amino acids in petioles attained their highest levels during mid and late pod-filling, while ¹⁴C-organic acids comprised 2% or less of the recovered radioactivity after pod initiation. The distribution of ¹⁴C-assimilates in the internode below the source leaf was similar to that found in petioles. A comparison of the above data to calculated C and N requirements for seed development suggests that ¹⁴C-amino acids derived from current photosynthesis and translocated from source leaves supply at least 12% to 48% of the seed N depending on the stage of pod-filling.     

Partitioning of C-photosynthate, and long distance translocation of amino acids in preflowering and flowering, nodulated and nonnodulated soybeans

The influence of stage of development (preflowering versus flowering) in nodulated and nonnodulated soybeans (Glycine max [L.] Merr. cv. Wells) on partitioning of ¹⁴C into assimilates following exposure of a soybean leaf to ¹⁴CO₂ by both steady-state and pulse-labeling techniques was studied. Blades on the second fully expanded leaf from the stem apex were exposed to ¹⁴CO₂. Radioactive assimilates were extracted from source leaf blades, petioles, and stems (both the path up and path down from source leaf), were separated into neutral (sugars), basic (amino acids), and acidic (organic acids, sugar phosphates) fractions by ion exchange chromatography. The basic fraction was further resolved using thin layer chromatography and the percentage of radioactivity recovered in each amino acid was determined.     

Metabolic Pathway of alpha-Ketoglutarate in Citrus Leaves as Affected by Phosphorus Nutrition

The uptake and metabolism of α-[5-¹⁴C]ketoglutarate by phosphorus-deficient and full nutrient (control) lemon (Citrus limon) leaves were studied over various time intervals. After 45 minutes in P-deficient leaves, the bulk of incorporated ¹⁴C appeared in organic acids and much less in amino acids, while in the control leaves, the ¹⁴C contents of organic and amino acids were equal. In P-deficient leaves, after longer incubation times the ¹⁴C content of organic acids and amino acids increased, while that of CO₂ and residue fractions remained low. In full nutrient leaves the ¹⁴C content of amino acids and organic acids decreased after longer incubation time and increased in the insoluble residue and CO₂. In full nutrient leaves the organic and amino acid metabolism were closely related and accompanied by protein synthesis and CO₂ release, while in P-deficient leaves an accelerating accumulation of arginine and citric acid was linked together with inhibition of protein synthesis and CO₂ liberation.     

Metabolism of Exogenous Malonate by Coffee Leaf Tissue

When [l-¹⁴C]-malonate was supplied to discs cut from matureleaves of Coffea arabica, ¹⁴CO₂ was released (approximately12% of the total CO₂ respired) and organic acids of the Krebscycle, uronic acids, sugars and amino acids became radioactive.There was no incorporation of MC into either lipids or phenoliccompounds. The formation of glucose from malonate has not beenobserved in other studies with plant tissues. The synthesisof labelled glucose together with an active pentose phosphatepathway that is stimulated by malonate explains the accumulationof radioactive phosphogluconate in the leaf discs. Tentativeproposals are made for pathways to account for the results obtained. Key words: Coffee leaves, Malonate metabolism, Pentose phosphate pathway     

Utilization of exogenously supplied primary precursors for essential oil synthesis in cymbopogon species

The relative efficiency of incorporation of the exogenously supplied primary precursors [2-¹⁴C]acetate, [U-¹⁴C]glucose and [U-¹⁴C]sucrose into essential oil was determined in the immature leaves of threeCymbopogon species(C. martinii,C. winterianus andC. flexuosus). Acetate was most efficiently incorporated into essential oil inC. winterianus andC. flexuosus, whereas glucose was the best precursor inC. martinii. The observations are consistent when expressed as radioactivity [Bq] of essential oil per leaf, as percent incorporation or moles of precursors utilized for essential oil synthesis. Thus, there is selectivity in the efficiency of precursor utilization for the oil synthesis inCymbopogon species.     

Glutamic Acid metabolism and the photorespiratory nitrogen cycle in wheat leaves: metabolic consequences of elevated ammonia concentrations and of blocking ammonia assimilation

The effects of methionine sulfoximine and ammonium chloride on [¹⁴C] glutamate metabolism in excised leaves of Triticum aestivum were investigated. Glutamine was the principal product derived from [U¹⁴C]glutamate in the light and in the absence of inhibitor or NH₄Cl. Other amino acids, organic acids, sugars, sugar phosphates, and CO₂ became slightly radioactive. Ammonium chloride (10 mm) increased formation of [¹⁴C] glutamine, aspartate, citrate, and malate but decreased incorporation into 2-oxoglutarate, alanine, and ¹⁴CO₂. Methionine sulfoximine (1 mm) suppressed glutamine synthesis, caused NH₃ to accumulate, increased metabolism of the added radioactive glutamate, decreased tissue levels of glutamate, and decreased incorporation of radioactivity into other amino acids. Methionine sulfoximine also caused most of the ¹⁴C from [U-¹⁴C]glutamate to be incorporated into malate and succinate, whereas most of the ¹⁴C from [1-¹⁴C]glutamate was metabolized to CO₂ and sugar phosphates. Thus, formation of radioactive organic acids in the presence of methionine sulfoximine does not take place indirectly through “dark” fixation of CO₂ released by degradation of glutamate when ammonia assimilation is blocked. When illuminated leaves supplied with [U-¹⁴C] glutamate without inhibitor or NH₄Cl were transferred to darkness, there was increased metabolism of the glutamate to glutamine, aspartate, succinate, malate, and ¹⁴CO₂. Darkening had little effect on the labeling pattern in leaves treated with methionine sulfoximine.